The overall aim of this procedure is to create a micro patterned, dual hydrogel cell culture system for neuronal studies. This is accomplished by first plating tissue implants such as embryonic, dorsal root ganglia on permeable cell culture inserts. Next, a micro pattern is created in a photo cross linkable hydrogel using a digital micro mirror device or DMD as a dynamic photo mask.
This is followed by the addition of the self assembling hydrogel like pure, which may contain a suspension of dissociated cells. Proper incubation time is necessary before cellular analysis. Ultimately, results can be obtained that show neuronal cell viability and neurite outgrowth through specific geometric configurations defined by the DMD.
The main advantage of this technique over existing methods is that a dynamic photo mass can be quickly created, modified, and projected from a distance to accommodate just about any substrate like permeable cell culture inserts. Though this method can provide insight into neural systems, it can also be applied to other systems which could benefit from specifiable geometric constraints such as cell signaling, cell migration, cell to cell contact, or morphogenesis to name a few. The DMD board UV lights guide and four times objective lens are all mounted vertically on a vibration isolation table.
An inverted microscope is placed below the objective lens such that the focus light reflected from the DMD can be visualized through the microscope. For additional details on the DMD setup, please see the accompanying written protocol. First, prepare the six well collagen coated cell culture inserts to receive X explan cultures coat the walls of the cell culture inserts with rain x, taking care not to apply rain X to the membrane itself.
Then add 1.5 milliliters of aian media to the inserts and incubate overnights at 37 degrees Celsius and 5%carbon dioxide. The next day after harvesting embryonic dorsal root ganglia from E 15 rat pups plate up to four DRGs onto each collagen coated.Six. Well insert and incubate for two hours at 37 degrees Celsius and 5%carbon dioxide to allow the DRG to adhere to the membrane.
After the incubation period, the DRGs will be ready for photo polymerization. A digital micro mirror device is a 1024 by 768 array of individual mirrors, which selectively reflects light based on mirror position. In this procedure, the DMD is used to pattern ultraviolet light onto photo CROs, linkable hydrogels, creating specifiable hydrogel geometries in a simple and rapid manner.
Though our DMD is a standalone unit, the device can also be integrated for use with many existing microscopes. First, retrieve the DRGX plants from the incubator, aspirate all excess liquid from the insert. Then add 500 microliters of polymerization medium to the inside of the insert.
Transfer the inserts with the DRGX plants onto a rain extre glass slide and place the slide underneath the DMD. Use the ALP three basic graphical user interface program to load the black and white image that will be used as the photo mask onto the DMD. In this case, a bifurcating shape was chosen to allow for implementation of neurite guidance systems using an inverted microscope and DI visible light source.
Align the tissue explan with the appropriate location on the photo mask using the light reflecting off the DMD as a guide. Next, switch the visible light source for a UV light source. Illuminate the polymerization medium inside the cell culture.
Insert until cross-linking of PEG is complete. Repeat for each DRG on the insert. Then wash each insert three times with sterile dcos phosphate buffered saline containing 1%penicillin streptomycin.
Finally, add 1.5 milliliters of growth media underneath the cell culture inserts and allow to equilibrate in a tissue culture incubator at 37 degrees Celsius and 5%carbon dioxide for 30 minutes. During the incubation period, sonicate pure matrix for 30 minutes. To solubilize the peptides dilute the supplied 1%solution to 0.15%in sterile water and supplement with one microgram per milliliter of soluble laminin.
After 30 minutes, remove the cultures from the incubator and aspirate all excess media from the insert. Then while visualizing the void under a microscope, aspirate media from inside the voided area in the PEG construct containing the DRGX explan pipette around one microliter of the pure matrix solution into the void around the X explan. Ensure that the pure matrix does not overflow the peg void.
Then pipette 1.5 milliliters of growth media underneath the insert and place in the tissue culture incubator at 37 degrees Celsius and 5%carbon dioxide for one hour. To ensure total ation after this time, replace the growth media and then return the cultures to the incubator. Growth media is replaced after every 48 hours of culture.
Examples of dual hydrogel constructs containing DRGX plants are shown here. The DRGX plants have been labeled for growth and proliferation markers. The polymerized PEG construct appears gray, DRG NEURITES labeled with beta three tubulin appear green and DPI stain cell nuclei as seen in blue.
Notice that cellular migration and retic extension is limited to the cell permissive region of the dual hydrogel construct. Dual hydrogel encapsulation is appropriate when using any self assembling gel, the photo cross linkable gel. In this case, PEG serves as a structural support for the geometric presentation of the self assembling gel.
For example, pure matrix or aris. The type of gel and choice of photo mask will depend on the particular desired application. Treat the walls of a polyester cell culture insert with rain X as previously demonstrated.
Transfer the insert onto a rain X coated slide and place under the DMD. Next, add 500 microliters of polymerization media to the insert. Load the appropriate mask on the DMD for this survival experiment.
A cylindrical presentation of pure matrix is used to aid in the imaging of cells. Expose the insert to UV light for 55 seconds to induce peg cross-linking after cross-linking. Wash the newly formed void with sterile DPBS and 1%pen strep.
Use a micro pipette or a sterile cotton tip applicator to remove all excess liquid from the void. Prepare pure Matrixx by Sonicate for 30 minutes, dilute to 0.15%in sterile water, supplemented with one microgram per milliliter of soluble laminin and 10%sucrose. Next, pellet the cells to be cultured by centrifugation.
Thoroughly aspirate the media from the cell pellet and re suspend the pellet at a concentration of approximately five times 10 to the three cells per milliliter in the 0.15%pure matrix solution. It is critical that all of the media is aspirated from the cell pellet as pure matrix begins self-assembly upon contact with salt solution. Next, pipette around one microliter of the supplemented pure matrix cell mixture into the peg void.
This volume is typically enough to fill the empty space without overflowing pipette 1.5 milliliters of growth media underneath the insert. Then place the pure matrix secondary hydrogel into the tissue culture incubator at 37 degrees Celsius and 5%carbon dioxide for one hour to ensure total gel lation after this time, aspirate the media and add fresh media underneath the insert. Taking care not to damage the mechanically weak pure matrix.
Return the cultures to the tissue culture incubator and change the media after every 48 hours of culture. Here are examples of live DRG neurons encapsulated inside a variety of dual hydrogel constructs. After 48 hours in growth medium, the cells were labeled with calcium am a live cell marker that appears green.
Here due to the dynamic nature of the DMD photo mask, the geometry a available for encapsulation is limited only by the dimensions and resolution of the optics. A single hydrogel encapsulation is appropriate where the cells can be examined inside of a photo cross linkable hydrogel true to polyester cell culture. Insert with rain X and place under the DMD on a rain X coated slide.
As before, pipette the cells to be cultured directly into the polymerization medium at the desired concentration and mix well to ensure homogenous distribution. Then pipette 500 microliters of the polymerization medium cell mix into the insert. Choose the appropriate mask and load it onto the DMD induce PEG cross-linking by exposure to UV light for 55 seconds.
For cell survival studies, a basic circular mask is used to represent a cylinder wash the insert three times with sterile DPBS and 1%pen strep, then pipette 1.5 milliliters of growth media underneath the insert and enough growth media on top to fully immerse the insert incubate 37 degrees Celsius and 5%carbon dioxide with media changes every 48 hours. Here is an example of live dissociated cortical neurons encapsulated inside peg. Live cells are labeled with calcium am and appear green while dead cells are labeled with ahy homodimer one and appear red.
Encapsulation in PEG is meant only as an example, as PEG does not represent an ideal environment for neural cells. The chosen geometry represents a simple means to study cell viability, but could be adapted to a wide range of structures. Thin film hydrogels can be used to generate specific patterns of restricted cell.
Again, treat to polyester cell culture insert with rain X as before and place onto a rain X treated slide. Then pipette 250 to 300 microliters of polymerization medium into the bottom of the treated cell culture insert. This volume is sufficient to just cover the bottom of the insert incubate at room temperature for 30 to 45 minutes to allow the medium to permeate the insert membrane aspirate excess medium from the insert with a micro pipette.
Then pipette sufficient amount of UV transparent oil into the bottom of the insert to completely cover the area. Incubate the insert at room temperature for 15 to 30 minutes, which is long enough for the oil to form a distinct layer above the polymerization medium. Saturating the insert membrane load an appropriate mask onto the DMD.
After placing the insert and slide under the DMD induce peg cross linking by exposure to UV light. Wash the insert three times with sterile DPBS and 1%pen strap after washing, transfer the insert to a six well tissue culture plate harvest and pellet the cells to be cultured. Aspirate the media and resuspend the cell pellet in growth media at the desired concentration between 10 to the four and 10 to the six cells per milliliter.
Then pipette sufficient volume of the cell suspension inside the cell culture insert to obtain the desired cell density. Add enough growth medium below the insert to completely maintain cell viability. Incubate 37 degrees Celsius and 5%carbon dioxide after 48 hours of incubation.
Wash the insert three times with sterile DPBS and 1%pen strep to dislodge any unaired cells. Then add fresh growth media to the wells. Return the plate to the tissue culture incubator and perform media changes every 48 hours.
Here the cell restrictive PEG has been polymerized as a thin film using a test pattern to selectively adhere dissociated cells to the collagen coated membrane of permeable supports. The cells were labeled after 48 hours of incubation. Live cells labeled with calc am appear green while dead cells are labeled with atherium homodimer one and appear red.
It can be seen that minimal cell aian occurs in the area containing their thin PEG film. This is an example of an undesirable result. Partial polymerization of PEG has rendered an unusable peg construct.
Improper polymerization can occur due to the presence of a meniscus in the pre polymer medium, insufficient amounts of polymerization, medium, insufficient UV exposure or improper focus of the optics. This is an example of a poor dual hydrogel 3D encapsulation of A-D-R-G-X plant culture. In this image, it can be seen that the neurites visible in green were able to grow outside of the patterned peg channels.
This often occurs if pure matrix overflows on top of the PEG portion during injection. Following this procedure. Other standard methods like patch clamping or immunohistochemistry can be performed to answer additional questions such as electrophysiology or phenotypic expression.
After watching this video, you should have a good understanding of how to use dynamic mask projection photolithography to specify the geometric presentation of single or dual hydrogel systems for the study of neuronal cell cultures.
